The role of gender and pain remains a major health care problem, and in preliminary studies on this topic, we evaluated the long-term effects of estradiol on gene expression in trigeminal neurons. The results constituted an unexpected discovery that estradiol upregulates prolactin (PRL) more than 40 fold in sensory neurons. Follow-up studies demonstrated that PRL and the PRL receptors (PRL-R) are expressed in sensory neurons of both female and male rats, and that application of capsaicin evokes PRL release from trigeminal sensory neurons. Furthermore, application of exogenous PRL significantly and acutely increases nociceptor responsiveness to capsaicin as measured by inward currents, CGRP exocytosis, accumulation of intracellular calcium levels, and nocifensive behavior. These preliminary data provide strong initial support for a completely new hypothesis of nociceptor regulation by an autocrine/paracrine system containing PRL. Based upon this hypothesis, PRL may serve as a novel hyperalgesic agent in both females and in males. We believe that this discovery has substantial scientific and medical implications, and is highly innovative from a conceptual perspective. Therefore, this project will characterize the mechanisms mediating prolactin effects in female and male rats and will directly test the hypotheses that PRL evokes a rapid increase in the responsiveness sensory neurons to noxious stimuli such as capsaicin. Our specific aims will: Specific Aim 1: Determine the effects of exogenous PRL on capsaicin- and inflammation-induced hyperalgesia/allodynia. Specific Aim 2: Determine the mechanisms by which PRL rapidly increases the responsiveness of trigeminal neurons to noxious chemical and thermal stimuli. Specific Aim 3: Characterize the stimuli that evoke PRL release in trigeminal sensory neurons from in vitro cultures and from acutely isolated and superfused peripheral terminals. The discovery that trigeminal sensory neurons express both PRL and PRLR, and that application of exogenous PRL significantly and rapidly sensitizes trigeminal nociceptors to noxious stimuli such as capsaicin, provides strong initial support for a completely new and innovative hypothesis of nociceptor regulation by an autocrine/paracrine PRL system, and compounds that block the PRL-R may serve as a novel class of analgesic drugs in gender dependent pain.